SDS Optic, EcoTech Complex, Block A, Głęboka 39, 20-612, Lublin, Poland.
Lukasiewicz Research Network - PORT Polish Center for Technology Development, Stabłowicka 147, 54-066, Wrocław, Poland.
Sci Rep. 2024 Feb 17;14(1):3978. doi: 10.1038/s41598-024-54590-z.
The expression of the HER2 (human epidermal growth factor receptor 2) protein in cancer cells is a well-established cancer marker used for diagnostic and therapeutic purposes in modern treatment protocols, especially in breast cancer. The gold-standard immunohistochemical diagnostic methods with the specific anti-HER2 antibodies are utilized in the clinic to measure expression level of the membrane-bound receptor. However, a soluble extracellular domain (ECD) of HER2 is released to the extracellular matrix, thus the blood assays for HER2 measurements present an attractive way for HER2 level determination. There is a need for accurate and validated assays that can be used to correlate the concentration of the circulating HER2 protein with disease clinical manifestations. Here we describe two monoclonal antibodies binding HER2 with a unique sequence of the complementarity-determining regions that recognize HER2 ECD. Development and validation of the sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the soluble HER2 in a variety of biological samples is also presented. The assay provides HER2 quantitation within a concentrations range from 1.56 to 100 ng/ml with sensitivity at the level of 0.5 ng/ml that meets the expectations for measurements of HER2 in the blood and tumor tissue samples. The method presents satisfactory intra- and inter-assay precision and accuracy for immunochemical quantification of biomarkers in biological samples. The utility of the generated monoclonal anti-HER2 antibodies has been confirmed for use in the precise measurement of HER2 (both cell-bound and soluble) in several types of biological material, including serum, solid tumor tissue, and cell culture medium. Additionally, the developed immunochemical tools have a potential for HER2 detection, not only in a wide range of sample types but also independently of the sample storage/pre-processing, allowing for comprehensive HER2 analysis in tissue (IHC), cultured cells (immunofluorescence) and blood (ELISA).
HER2（人表皮生长因子受体 2）蛋白在癌细胞中的表达是一种经过充分验证的癌症标志物，用于现代治疗方案中的诊断和治疗目的，特别是在乳腺癌中。临床中使用具有特异性抗 HER2 抗体的金标准免疫组织化学诊断方法来测量膜结合受体的表达水平。然而，HER2 的可溶性细胞外结构域（ECD）会被释放到细胞外基质中，因此血液中 HER2 测量的检测方法是确定 HER2 水平的一种有吸引力的方法。需要准确和经过验证的检测方法，以便将循环 HER2 蛋白的浓度与疾病临床表现相关联。在这里，我们描述了两种结合 HER2 的单克隆抗体，它们具有独特的互补决定区序列，可识别 HER2 ECD。还介绍了用于定量各种生物样品中可溶性 HER2 的夹心酶联免疫吸附测定（ELISA）的开发和验证。该测定法可在 1.56 至 100ng/ml 的浓度范围内定量 HER2，灵敏度为 0.5ng/ml，符合血液和肿瘤组织样品中 HER2 测量的期望。该方法在生物样品中免疫化学定量生物标志物的精密度和准确度方面均表现出令人满意的重复性和准确性。所产生的单克隆抗 HER2 抗体已被证明可用于精确测量多种类型的生物材料中的 HER2（包括细胞结合和可溶性），包括血清、实体瘤组织和细胞培养基。此外，开发的免疫化学工具具有用于 HER2 检测的潜力，不仅可以在广泛的样本类型中使用，而且可以独立于样本储存/预处理，从而可以在组织（免疫组化）、培养细胞（免疫荧光）和血液（ELISA）中进行全面的 HER2 分析。
